Pulmonary vascular resistance (PVR) in the fetus undergoes a dramatic decrease at birth to facilitate gas exchange during postnatal life. Exposure to higher oxygen tension at birth plays a major role in this adaptation. Endothelial nitric oxide synthase (eNOS) regulates the transition in PVR and is stimulated directly by oxygen and indirectly by an increase in oxidative phosphorylation and ATP release. Oxidative phosphorylation also generates superoxide (O27-) as a byproduct. However, the mechanisms that regulate mitochondrial oxidative stress in the fetal pulmonary arteries during this transition remain unclear. Failure of PVR to decrease at birth leads to persistent pulmonary hypertension of the newborn (PPHN), a condition associated with increased oxidative stress in pulmonary arteries. Identification of the sources of oxidative stress is critical to the development of specific therapies to correct the vascular dysfunction in PPHN. Studies in fetal lamb pulmonary artery endothelial cells (PAEC) demonstrated that exposure to ATP, a NOS agonist, or transition to postnatal oxygen tension stimulate the association of eNOS with the mitochondrial outer membrane protein, porin. NOS agonists increase mitochondrial NO release in fetal PAEC. We propose to investigate the novel hypothesis that targeting of eNOS to the mitochondrial outer membrane regulates the rate of oxidative phosphorylation and O27- generation in PAEC by targeted NO release during birth- related transition. The broad specific aims of the proposed studies are to (1) Investigate whether eNOS - mitochondrial interactions regulate the mitochondrial O27- during birth related transition in PAEC and to define the mechanism of this targeting and (2) determine whether eNOS-mitochondrial interactions regulate the endothelial functions critical to postnatal adaptation: synthesis of NO, PGI2 and endothelin-1 and postnatal pulmonary vasodilation. Studies will be done in PAEC and intact fetal lambs. Cell biology tools will be developed to (A) target eNOS to specific sub-cellular compartments in fetal PAEC and (B) to inhibit the interactions of eNOS with the client proteins involved in mitochondrial targeting - hsp90 and porin. The proposed studies will investigate a new regulatory mechanism for the mitochondrial oxidative stress during oxygen exposure at birth. Identification of this new source of oxidative stress in PAEC may lead to more specific therapies, such as mitochondrial targeted anti- oxidants to restore pulmonary vasodilation and oxygenation in PPHN. PUBLIC HEALTH RELEVANCE: Exposure of the fetal lung to oxygen at birth mediates the increase in pulmonary blood flow which is necessary to establish gas exchange during postnatal life. Oxygen increases the metabolism in mitochondria which in turn can increase free radical production. The proposed studies will investigate a new mechanism that regulates the formation of free radicals during this adaptation.